There are different known methods of producing peptides by biotechnological means. Since the stability of short polypeptide chains in microbial host cells is usually low, and since the free peptides may have a possible toxic effect on the host organism (for example antimicrobial peptides), most methods involve producing larger precursor proteins from which the peptide is excised after the precursor protein has been purified.
One possibility of obtaining a stable precursor protein comprises expressing a peptide together with a stable protein by way of a fusion protein. The properties of said fusion protein, which greatly influence subsequent work-up steps, are determined by the fusion partner largely independently of the peptide sequence, and are therefore readily controllable and suitable for producing peptides with different sequences.
WO 2008/085543 describes a special method of producing proteins and peptides with the aid of a fusion protein. This fusion protein comprises aside from the desired peptide sequence a fusion partner which ensures that the fusion protein exhibits an inverse phase transition behavior. This behavior firstly involves the fusion protein to be purified from the cellular context in a simple and inexpensive manner. Secondly, the fusion partner may likewise be removed in a simple and inexpensive manner, after the peptide has been removed by proteolytical cleavage. While a fusion protein may frequently be obtained with good yields, the peptide portion of the precursor protein is usually small, and the efficiency of the process is therefore suboptimal.
Another approach involves repetitive precursor proteins which comprise multiple copies of the desired peptide being recombinantly produced. WO 03/089455 describes the production of multimeric precursor proteins from which the desired peptide sequences which have antimicrobial properties are excised by acidic cleavage.
There are a number of further published approaches (examples: Metlitskaya et al. Biotechnol Appl. Biochem 39; 339-345 (2004); Wang & Cai Appl. Biochem and Biotechnol. 141; 203-213 (2007)), which were used for demonstrating that peptide sequences or families of peptide sequences may be produced by a particular method with the aid of repetitive precursor proteins. To some extent the use of special auxiliary sequences which are located between the repeats of the desired peptide sequences has been described. More specifically, anionic auxiliary sequences have been proposed which apparently reduce the harmful action of cationic antimicrobial peptide sequences within a repetitive precursor protein on the host cell (cf. for example WO 00/31279 and US 2003/0219854). While the precursor protein in this repetitive approach has a higher proportion of the desired peptide sequence than is the case with fusion proteins, the properties of the repetitive precursor proteins are greatly influenced by the sequence of the desired cationic peptide.
The inventors have no knowledge of any previous method involving the possibility of producing any peptide sequences with the aid of repetitive precursor proteins according to a simple, low-cost protocol which can be carried out in an efficient manner.
Various antimicrobial peptides have been described in the literature and are summarized in Reviews (Hancock, R. E. W. and Lehrer, R. 1998 in Trends in Biotechnology, 16: 82-88; Hancock, R. E. W. and Sahl, H. G. 2006 in Nature Biotechnology, 24: 1551-1557).
Fusion peptides, in which two active peptides are combined, are likewise described in the literature. Wade et al. report the antibacterial action of various fusions of Hyalophora cecropia cecropin A and the poison melittin (Wade, D. et al., 1992, International Journal of Peptide and Protein Research, 40: 429-436). Shin et al. describe the antibacterial action of a fusion peptide of Hyalophora cecropia cecropin A and Xenopus laevis magainin 2, consisting of 20 amino acids. Cecropin A consists of 37 amino acids and exhibits activity against Gram-negative bacteria that lower activity against Gram-positive bacteria. magainin 2 consists of 23 amino acids and is active against bacteria but also tumor cell lines. Compared with the fusion of cecropin A and melittin, this fusion exhibits a distinctly lower hemolytic activity with a comparable antibacterial action (Shin, S. Y. Kang, J. H., Lee, M. K., Kim, S. Y., Kim, Y., Hahm, K. S., 1998, Biochemistry and Molecular Biology International, 44: 1119-1126). US 2003/0096745 A1 and U.S. Pat. No. 6,800,727 B2 claim these fusion peptides, consisting of 20 amino acids, and variants of said fusion which, due to the substitution of amino acids, in particular of positively charged amino acids and hydrophobic amino acids, are more positively charged and more hydrophobic.
Shin et al., 1999, describe further developments of this cecropin A-magainin 2 fusion peptide. They demonstrated that the peptide having SEQ ID NO:6 had a lower hemolytic activity compared to the starting fusion but that the antibacterial activity with respect to Escherichia coli and Bacillus subtilis was not adversely affected (Shin at al. 1999 Journal of Peptide Research, 53: 82-90).